Parkinson's disease is related to a deficiency of complex I bioenergetic activity that may be etiologic and likely contributes to increased oxidative stress. The protein chemical work described here addresses the question inherent to other parts of this Udall proposal, i.e., "As Complex I activity is clearly reduced in Parkinson's Disease, based on several studies, is this altered functioning caused by disrupted assembly and/or altered stability, AND/OR is there post-translational oxidative modification of the complex that progressively inhibits functioning? Project 3 will address three Aims. Specific Aim 1: Complete technique development and establish hot spots of oxidative modification of Complex I. While we have made considerable progress in identifying the sites of oxidative damage in Complex I by peroxynitrite, we need to fully identify sites of modification by hydroxyl radical reaction. Also, we wish to develop an antibody that can detect tryptophan oxidation to facilitate identification of this modification. Specific Aim 2: Further characterization of the phosphorylation/dephosphorylation of Complex I. We have established that three subunits of Complex I are phosphorylated and there is evidence that these modifications affect activity. Recent work has identified a rare genetic form of Parkinson's disease due to mutation of a mitochondrial protein kinase. Thus, altered phosphorylation/dephosphorylation may be a factor in the altered Complex I functioning which is evident in PD. We plan to identify the phosphorylation sites on Complex I by mass spectrometry and evaluate the functional significance of each. Specific Aim 3: Analysis of Complex I in PD brains and the cell lines created by protofection technology in Dr. Bennett's project. We have developed the tools for analyzing the levels of Complex I, its activity and the assembly state in small amounts of mitochondria. We will use these techniques to evaluate the properties of Complex I in PD brains and cell lines, and appropriate controls. These same samples will also be analyzed for oxidative, post-translational modifications that could account for altered functioning of Complex I.